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PCT

WORLD INTELLECTUAL PROPERTY ORGANIZATION
Intcmadona] Bureau

INTERNATIONAL APPUCATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)

(51) InternatloDal Patent Classlficatfon ^ :
GOIN 33/558, 33/537, 33/53

(11) International Publication Number: WO 96/15454

(43) International Publication Date: 23 May 1996 (23.05.96)

(21) International Applicatkm Number: PCr/AU9S/00763

(22) International Filing Date: 16 November 199S (I6.1 1.95)

(30) Priority Data:
PM9500

16 November 1994 (16.1 1 .94) AU

(71) Applicants {for all designated States except US): AUS-

TRALIAN MEMBRANE AND BIOTECHNOLOGY
RESEARCH INSTTTUTE [AU/AUJ; 8 Australia Avenue,
Homcbush, NSW 2140 (AU). THE UNIVERSITY OF
SYDNEY f AU/AU); Sydney, NSW 2006 (AU).

(72) Inventors; and

(75) Inventors^Applicants (for US only): BRAACH-MAKSVYTIS.
Vijolcta, Lucija. Bronislava (AU/AU); 9 Darlcy Street,
Dulwich Hill, NSW 2203 (AU). CORNELL, Biuce, An-
drew (AU/AU); 58 Wycwnbe Road, Neutral Bay, NSW
2089 (AU). KING. Uoncl. George (AU/AU); 27 Beatrice
Street, North Rydc, NSW 2113 (AU). RAGUSE, Burkhard
(DE/AU); 2 Mttdies Road, St Ives, NSW 2075 (AU).

(74) Agent: F3. RICE & CO4 28A Montague Street, Balmain,
NSW 2041 (AU).

(81) Designated States: AL. AM. AT, AU, BB. BG. BR, BY, CA.
CH, CN, C2, DE, DK, EE, ES. FI. GB. GE. HU, IS, JP,
KE, KG, KP, KR, KZ. LK, LR, LS, LT, LU, LV, MD, MG,
MK. MN. MW. MX, NO. NZ, PL, PT, RO, RU, SD, SE,
SG, SI. SK. TJ, TM. TF, UA, UG. US, UZ, VN. European
patent (AT, BE, CH, DE. DK, ES, FR, GB, OR. IE. IT. LU.
MC. NL. PT. SE), OAPI patent (BF. BJ. CF. CO, CI, CM.
GA, GN. ML. MR, NE, SN. TD, TG), ARIPO patent (KE,
LS. MW. SD, SZ, UG).

Published

With international search report.

(54) Title: DETECnON DEVICE AND METHOD
(57) Abstract

The present invention provides an analyte detection device. The device comprises first and second zones, means to allow addition
of a probe to the first zone, means to allow addition of a sample suspected to contain an analyte and means to allow passage of the probe
from the first zone to the second zone. The first zone contains Ugands reactive with the analyte and the second zone includes a membrane
the impedance of which is dependent on the presence or absence of the probe and means to measure the impedance of the membrane. It
is prefened that the probe includes an ionophore, preferably gramicidin, TTie present invention also relates to methods of detecting the
presence of an analyte.

FOR THE PURPOSES OP INFORMATION ONLY

Codes used to identify States paity to the PCT aa the front pages of pamphlets publishing international
applications under the PCT.

Awtite

United Kingdon

MauritaDia

AiB&ilia

GeoiiiB

Malawi

Bartadw

Guinea

Hiftt

NcuKnanas

Belghtm

• GR

Greece

BurkinftFaso

Hm^aiy

Butpria

beland

NewZeatand

BCBIS

Italy

Poland

Bmil

Jipan

Foctugal

Belan»

Kaqra

Romania

CiiuKb

Kyiiysin

Rossian Fcdeiatioo

Ceotnl Africn Repttblk

Democniic People's Repoblit

Sudan

<^Koiea

Sweden

Swincrfond

Republic of Korea

Slovenia

COie d'lvotie

Kazakhsuj)

Skyvakia

Liechteouein

Senegal

ClUM

Sri Lanka

Chad

Czarhodovakit

Lnxcmbouis

Czech Repvl^c

Lwrto

Tapkisun

Geimtny

Monaco

Trinklad and Tob«go

Dciuiitffc

RepoUicof Mokiova

Ukraine

Spain

Madagascar

United States of America

Finland

Ma»

Uzbekistan

Franct

Mof^golia

Viet Nam

Gabon

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DETECTION DEVICE AND METHOD

The present invention relates to a devices for the detection of an
5 analyte in a sample and to a methods of detecting the presence of an anal3rte
in a sample.

Current technologies used in the diagnostic industry require laige
expensive equipment for the detection of analj^es. For example,
immunoassays require gamma-detectors» spectrophotometers, lasers, etc. and
10 DNA detection after PGR processes requires electrophoresis and absorption
methods, all of which depend on the specific probe used for signal
amplification.

A number of devices have been described in the literature which
have been designed for simple single-step assays and make use of area

15 separation to carry out the different reactions and washing steps required.
For example, antibody-based tests such as the pregnancy testing device
Tlearblue One-Step" by Unipath employ a wick to absorb urine which then
travels the length of a pen-like device. The hormone hCG is captured by the
first layer which contains mobile blue latex particles to which mAb has been

20 coupled. The urine flow carries the latex, and bound hCG, to a second area
containing inunobilised mAb recognising a second epitope site on the
hormone. Any hCG bound to the latex will be prevented from continuing
past the second area as evidence by a discrete blue line. In the absence of
hCG, the latex moves through to a third area and captured by immobilised

25 antl-Fc antibody. Other disposable devices use liquid-operated switch

(illustrated in Figure 12.7 Chapter 12 by A. P. H. Famsworth, in T^olecular
and Antibody Probes in Diagnosis" edited by M. R. Walker and R. Rapley,
John Wiley and sons. 1993). to carry out sequential steps in the ELISA-t3rpe
processes. In DNA-based technologies, a product for performing the

30 multiple steps required in PCR technology has been released which by

compartmentalising the different steps in a single disposable device offers
simplicity and reduction of cross-contamination of the PCR products.

In International Patent Application Nos. PCT/AU88/00273.
PCT/AU89/00352. PCT/AU90/00025. PCT/AU92/00132. PCT/AU93/0059D.

35 PCT/AU93/00620 and PCT/AU94/00202 there is disclosure of biosensors

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which can be used to detect analytes. The disclosure of these dociunents in
included herein by cross-reference.

It is believed that by adapting these biosensors and existing
diagnostic techniques improved detection devices and methods of detection
5 can be achieved.

Accordingly in a first aspect tiie present invention consists in an
anal}rte detection device comprising first and second zones, means to allow
addition of a probe to the first zone, means to allow addition of a sample
suspected to contain an analyte to the first zone, and means to allow passage

10 of the probe from the first zone to the second zone; the first zone containing
ligands reactive with the analyte and the second zone including a membrane
the impedance of wUch is dependent on the presence or absence of the
probe and means to measure the impedance of the membrane.

The means to allow addition of the probe and sample to the first

15 zone may be the same of different.

In a preferred embodiment of the present invention the probe
includes an ionophore, preferably gramicidin.

In a further preferred embodiment of the present invention the
membrane comprises a first and second layer of closely packed arrays of

20 amphiphilic molecules and a plurality of ionophores comprising a first and
second half menibrane spanning monomers* the first half niembrane
spanning monomers being provided in the first layer and the second half
membnme spanning monomers being provided in the second layer, the
second half membrane spanning monomers being capable of lateral diffusion

25 • within the second layer independent of the first half membrane spanning
monomers, the first half membrane spanning monomers being pravented
from lateral diffusion in the firat layer, and a second ligaind provided on at
least the second half membrane si»nning monomers, said second ligand
being reactive with the probe or a portion thereof, the binding of the probe to

30 the second ligand causing a change in the relationship between the first half
membrane spanning monomers and the second half membrane spanning
monomers such that the flow of ions across the membrane via the
ionophores is allowed or prevented, and measuring the impedance of the
membrane.

35 In yet another preferred embodiment the ligands in the first zone are

antibodies or binding fragments thereof.

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In a second aspect the present invention consists in a method of
detecting the presence of an analyte in a sample, the method comprising
contacting the sample with a carrier including a plurality of first ligands
reactive with the analyte to allow binding of the analyte to the carrier
5 ligands, contacting the carrier with a membrane comprising a first and
second layer of a closely packed array of amphiphilic molecules and a
plurality of ionophores comprising a first and second half membrane
spanning monomers, the first half membrane spanning monomers being
provided in the first layer and the second half membrane spanning

10 monomers being provided in the second layer, the second half membrane
spaiming monomers being capable of lateral diffusion within the second
layer independent of the first half membrane spanning monomers, the first
half membrane spanning monomers being prevented from lateral diffusion
in the first layer, and a second ligand provided on at least the second half

15 membrane spanning monomers, said second ligand being reactive with the
analyte or a portion thereof, the binding of the analyte to the second ligand
causing a change in the relationship between the first half membrane
spanning monomers and the second half membrane spanning monomers
such that the flpw of ions across the membrane via the ionophores is

20 allowed or prevented, and measuring the impedance of the membrane.

The first half membrane spanning monomer in the first layer may be
prevented from diffusing laterally using any of a nimaber of known
.techniques, however, it is presently preferred that the monomer and the
amphiphilic molecules each include or are decorated with at least one

25 moiety cross'linked with at least one corresponding moiety on another of
these molecules. Under appropriate stimulus, such as UV radiation or
ionising radiation, the cross-linkable moieties can be caused to polymerise
thereby resulting in the membrane being cross-linked in one la3rer.

The first half membrane spanning monomers may also be prevented

3 0 from difiusing laterally by selecting lipids for the first layer of the membrane
. which are ciystalline at room teinperature. This eliminates lateral diffusion
in the first layer.

In a farther preferred embodiment of the present invention the first
half membrane spanning monomers in the first layer are prevented from
35 diffusing laterally by fixing the first layer and the monomers therein to a

solid support. This may be achieved by providing groups on the amphiphilic

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molecules in the first layer and on the monomers therein which are reactive
with the solid support or with corresponding groups provided thereon.

In another prefered form of the invention a proportion of the
amphiphlic molecules are membrane spanning amphiphiles, the membrane
5 spanning amphiphiles being archeobacterial lipids or tail to tail chemically
linked bilayer amphiphiles. It is also preferred that the half membrane
spanning monomers are gramicidin monomers.

In yet another preferred embodiment the membrane includes a
plurality of third ligands attached to amphiphiles in the membrane,
10 preferably membrane spanning amphiphiles. These third ligands are

preferably prevented from diffusing laterally within the membrane. In the
device of the first aspect of the present invention these third ligands will be
reactive with probe or a portion thereof, whilst in the method of the second
aspect of the present invention they will be reactive with the analyte.
15 The ligands may be the same or different and are preferably selected

from the group consisting of polsrclonal or monoclonal antibodies, antibody
fragments including at least one Fab fragment, antigens, lectins, haptens,
chelating agents and dyes.

Tlie ligands are preferably attached to the ionophores and/or
20 membranes via linkers. Suitable linkers are set but iii PCT/AUgo/00025,
PCT/AU92/00132 and PCT/AU93/0Q509.

As will be reconised by those skilled in this field it preferable that
the membrane is attached to an electrode such that a reservoir exists
between the electrode and the membrane. Molecules and methods by which
25 this may be readily achieved are set out in PCT/AU92/00132 and

PCT/AU93/00509. As stated above the disclosures of these documents are
incorporated by cross raferance.

In a third aspect the present invention consists in an analsrte
detection device comprising:-
30 a membrane including ligands reactive with an analyte;

means to measure the impedance of the membrane; and
means to move an analyte bound to the ligands away from the
membrane without disrupting the binding of the ligands to the analyte;
wherein the movement of the analyte away from the membrane causes a
35 change in the impedance of the membrane.

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In a preferred embodiment of this aspect of the present invention the
anal)rte is bound to a carrier via a plurality of second ligands. Preferably the
carrier is a bead, or a charged or magnetic particle.

In a preferred embodiment the means to move the analyte comprises
5 an electric field, magnetic field or liquid flow.

In another preferred embodiment the membrane ligands are attached
to amphiphiles of the menibrane. movement of the analjrte causing
extraction of the ligands and attached amphiphiles from the membrane.

In yei another preferred embodiment the membrane ligands are
10 attached to ionophores within the membrane, movement of the analyte
caxising extraction of the ligands and attached ionophores from the
membrane. The ionophores are preferably gramicidin.

In a fourth aspect the present invention consists in a method of
determining the presence or absence of an analyte in a sample, the method
15 comprising adding the sample to the device of the first or third aspect of the
present invention and measuring a changing conductivity or capacitance of
the membrane.

In a preferred embodiment of the present invention the membrane is
as described in International Patent Application Nos. PCT/AU88/00273,
20 PCT/AU89/00352, PCT/AU90/00025, PCT/AU92/00132, PCT/AU93/00590,
PCT/AU93/00626 or PCT/AU94/00202.

In order that the nature of the present invention may be more clearly
understood preferred forms thereof will now be described with reference to
the following examples and figures in which:
25 Figure la shows a schematic representation of an embodiment of the

analyte detection device of the present invention.

Figure lb is an expanded view of Region B of Figiire la.
Figure 2 is a schematic representation of another embodiment of the
anal3rte detection device of the present invention.
30 Figure 3 is a schematic representation of an embodiment of the

method of the present invention.

Figures 4a and 4b are schematic representations of an embodiment of
the detection device of the present invention.

Figures 5a and 5b are schematic representations of another
35 embodiment of the detection device of the present invention.

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Figure 6 is a schematic representation of another embodiment of the
method and device of the present invention.

Figures 7a and 7b are schematic representations of an embodiment of
the present invention used in the detection of DNA,
5 Figure 8 shows the structure of linker lipid A*

Figure 9 shows the structure of linker gramicidin B.

Figure 10 shows the structure of membrane spanning lipid D.

Figure 11 shows the structure of biotinylated gramicidin E where

n = 5.

10 Figure 12 shows impedance measurements in Example 4.

Figure 13 shows impedance measurements in Example 5.
Figure 14 shows impedance measurements in Example 6.
As shown in Figure la the device 10 consists of two zones 12 and 14.
Zone 12 is provided with ligands 16 reactive with analyte 18. The probe 20
15 consists of a ligand 22 reactive vdth anal3rte 18 and a nmrker 24.

Zone 14 includes a sensing membrane 26. The membrane 26
comprises amphiphilic molecules 28 and ionophores 30 and 32. lonophore
30 includes ligand 34 which is reactive with marker 24.

In operation a sample suspected of containing analsrte 18 is added to
20 zone 12. Probe 20 is also added to zone 12. In the situation shown in Figure
la the analyte 18 binds to ligands 16 and is tbere^by immobilised. Ligand 22
of probe. 20 then also binds to analyte 18 and thereby immobilises the probe.
The probe 20 is therefore unable to travel to zone 14 including sensing
membrane 16. If the analyte is not present the probe 20 is then free to travel
25 to zone 14 and sensing membrane 26. Upon reaching sensing membrane 26
the marker 24 binds to ligand 34 causing a change impedance of the
membrane.

Figure 2 shows another embodiment of the analyte detecting device.
The analyte detecting device 40 comprisiBS zones 42 and 44. Zone 42

30 includes carrier 46 to which are attached ligands 48 reactive with anal]rte 50.
As shown in Figure 2 zone 42 also includes probe 52 which comprises
analyte 50 and marker SA (streptavidin). Zone 44 includes a sensing
membrane 54 and electrode 55. The sensing membrane 54 consists of
amphiphiles 56 ionophores 58 and ligands 60 and 62 which are attached to

35 ionophores 58 and amphiphiles 56 respectively.

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PCT/AU9SA)0763

In operation analyte 50 is added to zone 42. The analyte 50
competes with the analyte 50 component of probe 52 for binding to iigand
48. As is shown in Figure 2 this results in the release of probe 52 which
includes streptavidin. The probe 52 then passes to zone 44 and sensing
membrane 54. The streptavidin then binds with ligands 60 and 62 causing a
change in impedance of the sensing membrane 54. Clearly, if the sample
added did not include analyte 50 probe 52 would not be released and the
streptavidin would not reach the sensing membrane 54.

Figure 3 shows an embodiment of the method of the present
invention. The method involves the use of a sensing membrane 70
comprising aniphiphiles 72 and ionophores 74 and 76 and electrode 71.
Ligands 78 and 80 reactive with analyte 82 are attached to ionophores 76 and
amphiphiles 72 respectively. A carrier bead 86 provided with a plurality of
ligands 84 reactive with analyte 82 is also provided. The binding at the
analyte 82 which is attached to the carrier bead 86 via ligands 84 to ligands
78 and 80 causes a change in impedance of the membrane 70.

Figure 4 shows schematically the operation of an embodiment of the
device of the present invention. As shown in Figure 4a an analsrte 92 is
bound to a carrier 90 via ligands 91. A sensing membrane 94 comprising
amphipfhiles 95 and ionophores 96 and electrode 99is also provided. The
analyte 92 is bound to the sensing membrane 94 via ligands 93. In Figure 4b
the carrier 90. and thereby analyte g2;ha5 been moved away from the
sensing membrane 94. This may be achieved by the application of force due
to an electric field, magnetic field or liquid flow. The movement of the
particle 90 causes the extraction of a segment 97 of the sensing membrane
95. This resiilts in an increased ability for ions to pass through the
membrane thereby resulting in a change in impedance of the sensing
membrane 94.

Figure 5 shows an alternate embodiment to that shown in Figure 4.
In this arrangement movement of the carrier 90 away from the membrane
results in extraction of ionophores 98 from the membrane. The removal of
these ibnophores will result in a decrease in the ability of ion to pass
through the membrane and therefore result in a change in impedance of the
membrane.

Figure 6 shows an alternate embodiment to that shown in Figure 3.
In this embodiment a sensing membrane 120 comprising amphiphiles 122,

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ionophores 124 and ligands 126 reactive with analyte 130 is provided. An
electrode 121 is also provided. A carrier bead 128 provided with a plurality
of ligands 132 reactive with analyte 130 and a plurality of ionophores 134 is
also provided. The binding at the analyte 130 to ligands 126 and 132 results
5 in the insertion of ionphores 134 into the membrane 120 thereby causing a
change in impedance of the membrane 120.

Figure 7 shows a schematic representation of the detection of DNA.
Figure 7a shows the sensing membrane 100 composed of amphiphiles 102
and ionophores 104 and 106 and electrode 101. Streptavidin (SA) is

10 attached to the amphiphiles 102 and ionophores 106 via linkers 108 and 110
respectively. As shown in Figure 7b biotin 114 on DNA 112 binds to the
streptavidin which causes a gating of the membrane 100 resulting in a
change of impedance of the membrane 100.

As will be appreciated, the representations in Figure 7 are an

15 embodiment of the second zone of the device of the present invention in
which the biotin labelled DNA functions as the probe.

As will be recognised by those skilled in the art the present
invention has general applicability, for example:-
20 1. Generic homogenous capillary/colmnn sensor * use vdth Ab-Ag-Ab
sandwich

a) DIRECT ASSAY: Sample added to assay device. Capillary action
drives the sample into contact with Ab labelled with probe. Further travel
enables the Ag-Ab complex to bind to a second Ab inmipbilised on capillary

25 wall which captures Ag*Ab complex. In the absence of anal}rte, the second
Ab labelled with probe diffuses to the biosensor membrane where it elicits a
change of impedance (Figure 1).

The detection probe may be an3rthing that upon incorporation into or
accrual onto the bilayer membrane elicits an impedance change (whether

30 increase or decrease in signal). Examples of detection probes include

streptavidin, gramicidin, gramicidin/detergent (e.g. SDS, octylglucoside)
aggregate, gramicidin/vesicle, gramicidin/polystyrene beads, etc. Where the
probe is streptavidin. the membrane would contain biotinylated gramicidin:
if the probe contains gramicidin, the membrane would initially contain no

35 gramicidin. Where the probe is an antibody the membrane would contain a
gramicidin-hapten or gramicidin-antigen conjugate.

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b) COMPErmVE ASSAY: as described for a) except that the
sandwich is preformed between Ab-Ag or Ag analogue-Ab. As the sample is
introduced, the analyte in the sample competes off the labelled second Ab,
eliciting an impedance change at the biosensor membrane.

2. Streptavidin Sensor

Bead column comprised of e.g. polystyrene beads coupled to Ab. A
covalently linked conjugate of analyte or analyte analogue and streptavidin
are bound to the Ab. When sample is introduced containing the anal)rte, the
analyte competes off the SA/analyte conjugate, releasing SA. SA binds to
biotinylated gramicidin in the biosensor membrane changing the impedance
signal (Figure 2). Can also be used in capillaiy mode as described in 1.
above. It will also be readily appreciated by persons skilled in the art that
such an arrangement may be used with labels (probes) other than SA. For
example SA could be replaced with a hapten and the gramicidin in the
membrane would, as opposed to being biotinylated, would have bound
thereto a receptor for the hapten.

3. Methods of detecting Ab-Ag-Ab sandwich involving Ab-bead
conjugates

a) LATERAL SEGREGATION; Ab-coated beads capture sample analyte.
Sandwich complex is completed with Ab linked to gramicidin and
membrane spanning lipid, causing lateral segregation of channels which
results in impedance change (see Figure 3).

b) LARGE PARTICLES INDUCING CURRENT LEAKS: Ab-coated large
beads capture sample analyte. Sandwich complex is completed with Abs
linked to membrane components which arei themselves cross-Unked into
domains (membrane may contain no chaimels). Application of liquid flow
at high velocity, removes a section of the biosensor membrane via the
domains resulting in electrical leakage. iSee Figiu^ 4.

c) LARGE PARTICLES REMOVING ION CHANNELS: Ab-coated laige
beads capture sample analyte. Sandwich complex is completed with Ab
linked to gramicidin in the biosensor membrane. Application of liquid flow
at high velocity, removes the gramicidin from the biosensor membrane
resulting in turning "off the electrical signal. See Figure 5.

d) MAGNETIC PARTICLES INDUCING CURRENT LEAKS: Ab-coated
charged magnetic beads capture sample anal)^e. Sandwich complex is
completed with Abs linked to membrane components which are themselves

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cross-linked into domains (contains no channels). Application of electric
field, or magnetic field, removes a section of the biosensor membrane via the
domains resulting in electrical leakage. See Figure 4.
e) MAGNETIC PARTICLES REMOVING ION CHANNELS: Ab-coated
5 charged magnetic beads capture sample analyte. Sandwich complex is
completed with Ah linked to gramicidin in the biosensor menabrane.
Application of electric field, or magnetic field, removes the gramicidin from
the biosensor membrane resulting in turning "ofP the electrical signal. See
Figures.

10 fj BEAD INSERTING ION CHANNELS INTO MEMBRANE: Ab-coated
beads coated with gramicidin channels capture sample anal)rte. Sandwich
complex is completed with Ab linked to components in the membrane
(contains no ion channels). The proximity oiF tfie beads to the surface allows
for the insertion of gramicidin chaimels on the beads into the membrane,

15 resiilting in conduction across the membrane. See Figure 6.
4. Method of detecting PCR products

Sample DNA is amplified using known PCR technology to generate
biotinylated-DNA. Biotinylated-DNA is passaged to biosensor membrane
containing SA linked to either gramicidin only or gramicidin and membrane

20 spaiming lipids, to directly or using lateral segregation, respectively, turn
"off the membrane. See Figure 7.

Example 1: Preparatioii of sensing membrane

25 The structure of linker lipid A is shown in figure 8; the structure of

linker gramicidin B is shown in figure 0; the structure of membmne
spanning lipid D is shown in figure 10; the structure of biotinylated
gramicidin E used, where n=: 5, is shown in figure 11.

Thus, a glass slide or plastic suppport is evaporatively coated with a

30 50 angstrom chromium adhesion layer, followed by a 2000 angstrom layer of
gold. The gold coated substrate is placed in an ethanolic solution containing
linker lipid A (300 ul of 10 mM solution in ethanol), 2«2'*ethanoI disulfide
(200 ul of a 10 mM solution in ethanol), linker gramicidin B (100 ul of a 0.01
mg/ml solution in ethanol), membrane spanning lipid D (225 ul of a 1 mM

35 solution in ethanol) and ethanol (50 ml). The gold coated substrate should
preferably be placed into this solution within five minutes of preparation.

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* 11

The gold coated substrate is left in this solution for 60 minutes, and then
rinsed with ethanoL The gold coated slide is then assembled in an electrode
holder such that an electrode is defined » that for the current examples has an
area of approximately 16 mm^. Then 5ul of a solution of l»2-di(3RS,7R,llR-
5 ph5rtanyl)-sn-gl3rcero-3-phosphocholine and l,2-di(3RS,7R,llR-

ph)rtanyI)glycerol in a 7:3 ratio, 14 mM total lipid concentration in ethanol is
added to the surface of the gold electrode and then rinsed with two washes
of 500 ul of phosphate buffered saline (PBS), leaving 100 ul PBS above the
electrode surface. The amount of PBS left above the electrode is preferably

10 less than or equal to 100 ul. A counter electrode, typically silver, is

immersed in the PBS solution; and the counter electrode and the sensing
electrode are connected to an impedance bridge. A DC offset of -300 mV is
applied to the sensing electrode during the AC measurement. The electrode
assembly is equilibrated to 35**C. This forms the sensing membrane for the

15 case when a probe is used that increases the conductance of the membrane.

Example 2: Preparation of probe solution

A solution of linker gramicidin B (luM) and sodium dodecylsulfate
20 (lOuM) in PBS is sonicated in a bath sonicator for 20 minutes. This solution
may be stored for at least 12 months at 4*'C. Although the gramicidin with
sodium dodecylsulfate is stable in aqueous solution, the gramicidin
incorporates readily into sensing membranes and produces conducting ion
channels. This change in conduction can be monitored using impedance
25 spectroscopy.

Example 3: Preparation of an avidin coated solid support

Pol3rst3rrene wells, as used in the preparation of ELISA tests, are
30 treated with a solution of avidin (1 mg/ml) in PBS for 60 minutes, and then
rinsed with PBS three times, drained, and then filled with 200 ul of PBS. The
pol]rst3aene wells are now coated with avidin.

Example 4: Sensing of small analyte - ie. biotin

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Two polystyrene wells coated with avidin are prepared as described
in example 3 - well A and well B. To well A, 5ul of a test solution containing
the anal3rte biotin (1 mM in PBS) is added and is mixed for 3 minutes. To
well B» 5ul of a test solution containing no biotin is added and mixed for 3
5 minutes. To both wells A and B, 2.5 ul of the probe solution prepared in

example 2 is added and mbced for 5 minutes. It is fotmd that in the presence
of the analyte (ie.biotin), the biotin is complexed to the receptor bound to
the solid support, in this case avidin, hence preventing the biotinylated
gramicidin E from complexing with the avidin on the solid support ie. the

10 biotinylated gramicidin E probe remains in the PBS solution. In the case
where no analyte (ie. biotin) is present in solution the receptor sites of the
avidin remain uncomplexed and the biotinylated gramicidin E probe is
complexed to the solid support ie. the biotinylated gramicidin E is removed
from the solution. Next, 100 ul of the solutions from well A and bom well B

15 are added to two separate sensing membranes and the conduction of the
membrane is monitored using impedance spectroscopy. Figure 12 shows
that the drop in impedance caused by addition of the solution from well A is
larger and faster than the drop in impedance caused by addition of solution
of well B. Thus the presence or absence of the biotin analyte can be

20 detected. The amount of biotin in the test solution will obviously determine
the number of binding sites that the biotin occupies on the receptor on the
solid support, which will in tum determine the number of probe molecules
left in solution. The rate of change of the impedance properties of the
membrane due to the probe will therefore be proportional to the anal}rte

25 concentration. Alternatively, when the number of probe molecules is

limited, the absolute number of probe molecules that affect the membrane
may be used to determine the concentration of analyte. It is known in the art
that it is possible to measure the conductance of a single gramicidin ion
channel in black lipid membranes. It will be appreciated by those skilled in

30 the art that the receptor bound to the solid support may be a receptor such as
an antibody specific towards an analyte, and that the gramicidin may have
an analogue of the anaMe attached such that the gramicidin can bind to the
attached receptor via the attached analyte analogue.

35 Example 5: Sensing oflarge analyte - ie. biotinylated BSA

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Two polystyrene wells coated with avidin are prepared as described
in example 3 - well C and well D. To well C, 5ul of a test solution containing
the analyte biotinylated bovine serum albumin (BSA) (1.3 mg/ml in PBS) is
added and is mixed for 10 minutes. To well D, 5ul of a test solution
5 containing no biotinylated BSA is added and mixed for 10 minutes. To both
wells C and D, 2.5 ul of the probe solution prepared in example 2 is added
and mixed for 10 minutes. It is expected that in the presence of the analjrte
ie.biotinylated BSA. the biotinylated BSA is complexed to the receptor
bound to the solid support in this case avidin. hence preventing the

10 biotinylated gramicidin E from complexing with the avidin on the solid

support ie. the biotinylated gramicidin E probe remains in the PBS solution.
In the case where no analyte (ie. biotinylated BSA) is present in solution the
receptor sites of the avidin remain uncomplexed and the biotinylated
gramicidin E probe is complexed to the solid support ie. the biotinylated

15 gramicidn E is removed firom the solution. Next, 100 ul of the solutions from
well C and from well D are added to two separate sensing membranes and
the conduction of the membrane is monitored using impedance
spectroscopy. Figure 13 shows that the drop in impedance caused by
addition of the solution from well C is larger and faster than the drop in

20 impedance caused by addition of solution of well D. Thus the presence or
absence of the biotinylated BSA analyte can be detected.

Example 6: Senring of lai*ge analyte - ie. ferritin

25 The polystyrene wells coated with an anti-ferritin antibody from a

conunercially available EUSA kit for ferritin (Bioclone Australia Pty. Ltd.,
Marrickville NSW 2204. Elegance Amplified Elisa iSystem, Cat No. FEA-96)
was used. To one well (well E), 200 ul of 500 nM ferritin was added, to
another well (well F) 200 ul of PBS without the ferritin analyte was added.

30 Both wells were mixed for six minutes and then washed with three times
400 ul PBS. Then 200 ul of biotinylated anti-ferritih antibody solution from
the EUSA kit was added to each well and mixed for 3 minutes. The wells
were rinsed with three times 400 ul PBS and 200 ul of 0.025 mg/ml of avidin
in PBS was added to both wells and mixed for 5 minutes. The wells were

35 rinsed with three times 400 ul PBS and 200 ul of PBS was left in both wells.
To both wells. 2.5ul of biotinylated gramicidin E/sodiiun.dodecylsulfate

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probe solution prepared in example 2 was added and mixed for 5 minutes.
Next, 100 ul of the solution from weU E and from well F were added to two
sensing membranes, as prepared in example 1. The change in impedance
due to the addition of the probe solution was monitored by impedance
5 spectroscopy. Figure 14 clearly shows that there is a larger and faster drop
in impedance due to the probe solution in the absence of ferritin from the
test solution than in the presence of ferritin in the test solution. As will be
readily appreciated the rate of change and the amplitude can be used to
determine the concentration of the ferritin in an analjrte sample.

10 As will be apparent from the above description the present invention

describes devices and methods which can be incorporated into current
detection methods for antibody or DNA-based technologies. The invention
uses the sensing membranes material described in various patents (e.g.
FCT/AU88/00273. PCT/AU89/00352, PCT/AU90/0D025. PCT/AU92/00132,

15 PCT/AU93/00590, PCT/AU93/00820 or PCT/AU94/00202) as the detection
material. The sensing membrane can be incorporated into single*step
devices or used in conventional multi*step processes to replace the enzyme,
chemiiuminescent, fluorescent, or radiolabelled, probes currently used for
the detection of end-product The type of probes which can be attached to

20 molecules whicli are used in the final step of antibody or DNA-based

technologies include any species which can cause a change in conduction
through the membrane.

For example, probes such as ion channels can insert themselves into
the membrane and allow ion flow across an insulting membrane. Other

25 probes can cause leaking paths across insulating membranes by specifically
binding to sites on the membrane and inducing either phase separation or
aggregation of molecules, solubilising the membrane, or removing a section
of the membrane.

Other probes may reduce the ion flow across the channel by

30 interacting with ion channels already present in the membrane. For
example, using streptavidin or avidin as the probe for interaction with
membranes containing biotinylated gramicidin will reduce ion flow across
the membrane.

The effect on the membrane can be amplified by the use of

35 multiprobes, such as latex or pply5t}rrene beads with a large mmniber of

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streptavidins bound to them to reduce ion flow, or abound to ion channels to
include ion flow across the membrane.

The advantages of the sensing membrane as detection mechanism in
antibody or DNA-based technologies is the speed and simplicity of the
5 readings. Ion flow changes can be measured by impedance changes at a
variety of frequencies or at a single frequency. Single-channel
measurements of, for example, gramicidin, are routinely carried out using
black lipid membranes, and offer the potential for extremely sensitive
measurements. Impedance measuietnehts require simple computational

10 equipment which can also be reduced in size to portable dimensions.

Reagents are simplified and do not rely on colour changes or light-emitting
species for detection.

It will be appreciated by persons skilled in the art that numerous
variations and/or modifications may be made to the invention as shown in

15 the specific embodiments without departing from the spirit or scope of the
invention as broadly described. The present embodiments are, therefore, to
be considered in all respects as illustrative and not restrictive.

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Claims:

1. An analyte detection device comprising first and second zones,
means to allow addition of a probe to the first zone, means to allow addition
of a sample suspected to contain an analyte and means to allow passage of

5 the probe firom the first zone to the second zone; the first zone containing
ligands reactive with the anal]rte and the second zone including a membrane
the impedance of which is dependent on the presence or absence of the
. probe and means to measure the impedance of the membmne.

2. An analyte detection device as claimed in claim 1 in which the probe
10 includes an ionophore.

3. An analyte detection device as claimed in claim 2 in which the
ionophore is gramicidin.

4. An analyte detection device as claimed in claim 1 in which the
membrane comprises a first and second layer of a closely packed array of

15 amphiphilic molecules and a plurality of ionophores comprising a first and
second half membrane spanning monomers, the first half membrane
spanning monomers being provided in the first layer and the second half
membrane spanning monomers being provided in the second la3^r, the
second half membrane spanning monomers being capable of lateral diffusion

20 within the second layer independent of the first half membrane spanning
monomers, the first half membrane spanning monomers being prevented
from lateral diffusion in the first layer, and a second ligand provided on at
least the second half membrane spanning monomers, said second ligand
being reactive with the probe or a portion thereof, the binding of the analyte

25 to the second ligand causing a change in the relationship between the first

half membrane spanning monomers and the second half membrane spanning
monomers such that the flow of ions across the membrane via the
ionophores is allowed or prevented, and measuring the impedance of the
membrane.

30 5. An analyte detection device as claimed in any one of claims 1 to 4 in
which the ligands in the first zone are antibodies or binding fragments
thereof.

6. An anal5rte detection device as claimed in claim 4 or claim 5 in
which a proportion of the amphiphlic molecules are membrane spanning
35 amphiphiles, the membrane spanning amphiphiles being archeobacterial
lipids or tail to tail chemically linked bilayer amphiphiles.

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7. An analyte detection device as claimed in any one of claims 4 to 6 in
which the half membrane spanning monomers are gramicidin monomers.

8. An anal)rte detection device as claimed in any one of claims 4 to 7 in
which the membrane includes a plurality of third ligands reactive with the

5 probe or a portion thereof attached to amphiphiles in the membrane.

9. An analyte detection device as claimed in claim 8 in which the
amphiphiles are membrane spanning amphiphiles.

10. An analyte detection device as claimed in claim 8 or claim 9 in
which the third ligands are prevented from diffiising laterally within the

10 membrane.

11. An analyte detection device as claimed in any one of claims 4 to 10
in which the membrane is attached to an electrode such that a reservoir
exists between the electrode and the membrane.

12. A method of detecting the presence of an analyte in a sample, the

15 method comprising contacting the sample with a carrier including a plurality
of first ligands reactive with the analyte to allow binding of the analyte to
the carrier ligands, contacting the carrier with a menibrane comprising a first
and second layer of a closely packed array of amphiphilic molecules and a
plurality of ionophores comprising a first and second half membrane

20 spanning monomers, the first half membrane spanning monomers being
prpvided in the first la3^r and the second half membrane spanning
monomers being provided in the second layer, the second half membrane
spanning monomers being capable of lateral diffusion within the second
layer independent of the first half membrane spanning monomers, the first

25 half membrane spanning monomers being prevented from lateral diffusion
in the first layer, and a second ligand provided on at least the second half
membrane spanning monomers, said second ligand being reactive with the
anal3rte or a portion thereof, the binding of the analyte to the second ligand
causing a change in the relationship between the first half membrane

30 spanning monomers and the second half membrane spanning monomers
such that the flow of ions across the membrane via the ionophores is
allowed or prevented, and measuring the impedance of the membrane.

13. A method as claimed in claim 11 in which a proportion of the
amphiphlic molecules are membrane spanning amphiphiles, the membrane

35 spanning amphiphiles being archeobacterial lipids or tail to tail chemically
linked bilayer amphiphiles.

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14. A method as claimed in claim 12 or claim 13 in which the half
membrane spanning monomers are gramicidin monomers.

15. A method as claimed in any one of claims 12 to 14 in which the
membrane includes a plurality of third ligands reactive with the analyte

5 thereof attached to amphiphiles in the membrane.

16. A method as claimed in claim 15 in which the amphiphiles are
membrane spanning amphiphiles.

17. A method as claimed in claim 15 or claim 16 in which the third
ligands are prevented from diffusing laterally within the membrane.

10 18. A method as claimed in any one of claims 12 to 17 in which the
membrane is attached to an electrode such that a reservoir exists between
the electrode and the membrane.
19. An analjrte detection device comprising;*

a membrane including Bgands reactive with an analyte;

^5 means to measure the impedance of the membraiie; and

means to move an analyte bound to the ligands away tem the
membrane without disrupting the binding of the ligands to the analyte:
whetein the movement of the analyte away from the membrane causes a
chcmge in the impedance of the membrane.
20 20. An analyte detection device as claimed in claim 10 in which the
analyte is bound to a carrier via a plurality of second ligands.

21. An analyte detectioii device as claimed in claim 20 in which the
carrier is a bead, or a charged or magnetic particle.

22. An analyte detection device as claimed in any one of claims 19 to 21
25 in which the means to move the analyte comprises an electric field,

magnetic field or liquid flow.

23. An analjrte detection device as claimed in any one of claims 10 to 22
in which the membrane ligands are attached to amphiphiles of the
membrane, movement of the analyte causing extraction of 4e ligands a^^

30 attached amphiphiles from the membrane.

24. An analyte detection device as claimed in any one of claims 19 to 22
in which the membrane ligands are attached to ionophores within the
membrane, movement of the analyte causing extraction of the ligands and
attached ionophores from the membrane.

35 25. An analyte detection device as claimed in claim 23 in which the
ionophores are gramicidin.

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26. A method of detecting the presence of an anal3rte in a sample
comprising adding the sample to the detection device as claimed in any one
of claims 1 to 11 or 19 to 25 and measuring the impedance of the membrane.

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SUBSTITUTE SHEET (RULE 26)

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SUBSTITUTE SHEET (RULE 26)

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3/14

SUBSTITUTE SHEET (RULE 26)

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4/14

SUBSTITUTE SHEEt(RULE26)

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PCT/AV95/00763

5/14

SUBSTITUTE SHEET (RULE 26)

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6/14

SUBSTITUTE SHEET (RULE 26)

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PCT/AV95AH)763

7/14

SUBSTITUTE SHEET (RULE 26)

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8/14

Linker Lipid A

()

I i

Fig. 8

SUBSTITUTE SHEET (RULE 26)

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9/14

GRAMICIDIN-0

Linker Gramicidin B

Fig. 9

SUBSTITUTE SHEET (RULE 26)

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10/14

Membrane Spanning Lipid D

Membrane Spanning Lipid C

FIGURE 10

SUBSTITUTE SHEET (RULE 26)

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11/14

m NH

GRAMICIDIN'

n=U3,4,5,6,7,8

Biotinylated Gramicidin E
Fig. 11

SUBSTITUTE SHEET (RULE 26)

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12/14

noBiotin
WELLB

WELL A

500000

;^ 100000

u>
9

10000

300

600 900
TIME (seconds)

1200

1500

FIGDRE 12

SUBSTITUTE SHEET (RULE 26)

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13/U

500000

N 100000
in

10000

500

biolin-BSA

well C

no biotbi'BSA

well D

700 900 1100

TIME (seconds)

1300

1500

FIGURE 13

SUBSnrUTE sheet (rule 26)

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14/14

+ Ferritin

well E

--A-- no Ferritin

well F

500000

:£ 100000
o

10000

Addtn of probe

W I M I H I I I Hin i lHll i ii i i iii

^^^^^^

wellE

wellF

-L

500 700

900 1100 1300 1500
Ti ME (seconds)

FIGDRE 14

SUBSTITUTE SHEET (RULE 26)

FNTERNATIONAL SEARCH REPORT

A- CLASSIFI CATION OF SUBJECT MATTER

Int CP- COIN 33/558, 33/537. 33/53

Imemational Application No.
FCT/AU 95/00763

to imeniational Patent ri:>ssificatioii OPC) or to both iiatkma! d««ifir^n« .^a mn
B. FIELDS SEARCHED

Atoimum documentaticm searched (classificaticm system foilowed by classiftcaUra symbols)
IPC GOIN 27/02 33/53 33/537 33/558

UiUCWENT : membmc, impedance, iiiq)edi^ «rawiisca;

DOCUMENTS CONSIDERED TO BE RELEVANT

Categcny*

Citation of document, with indicat ion, where ^ropriate. of the relevant

EP, A. 342382 (GENERAL ELECTRIC COMPANY) 23 November 1989"
claim 1

US, A. 4713347 (MITCHELL. D JI. & R.M.) 15 December 1987
claim 1

1 I ''"rtlw^^ocumeniiire lilted m the

Relevant to Claim No.
1 to26

1-26

|~| Seepunitfkniilytnnex

•A"
•E-
•L"

•O"
•P*

Spedal categories of cited docianaits:

document dcOning the general stale of the art which is
not consideied to be of partioilarrelevaDce
earlier document but published on or after the
international fiUi^ date

doeumcm >»hich may tfaiow doubts on priority cla^
or which is cited to establish the publicatioa date of
another citation or oiher spctM reason (as q)ecified)
document relaziqg to an oral disclosure, use.
odubition or other means

<tocnmem publidied prior to the intemstionaj filing
dale but later tfian the prioritvd^e^^ed

"X*

later document puUisfacd after the intcniatioiuU filing date or
priority date and not in confiict with the plication but cited to
understand the principle or theory underlying the invention
documait of particular relevance; the claimed invention cannot
be considered novd or cannot be ccmsidered to involve an
inventive stq> when the document is taken alone
documcBt of particular relevance; the claimed invention cannot
be ccmsidered to involve an inventive step when the document is
combined with one or more other such documents, such
CQaibinaticm being obvious to a person skilled in the art
document member of the same patent family

Date of the actual completion itf the international search
20FebroBry 1996

Name and mailing address of the ISA/AU
AUSTOAUAN INDUSTRIAL PROPERTY ORGANISATION
POBOX200
WODEN ACT 2606

AUSTRALIA Facsimile No.: (06) 285 3929
Form PCT/lSA/210 (second sheet) (July 1 992) copvak

Date of mailing of the intonatiQoal search report

29 February 1996 j

Authorized ofiicer
f I

^^BEL TYSON ^1^^
TdephQneNo.:(06>283 2<

^ 1

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